201113024 六、發明說明： 【發明所屬之技術領域】 本發明係關於使用硫酸軟骨素促進骨髓細胞分化，且更特別 地’係關於經輻射線處理之硫酸軟骨素用於促進骨髓細胞分化之 用途。 【先前技術】 近年來’已有文獻認為’醣胺素(GAGs)中之硫酸軟骨素可能 • 具有治療退化性關節炎的潛力（參見，例如，CordobaF，NimniME. 0你⑽呦rto 2003; 11 ⑶:228-230 ;及 Lippiello L. (¾加2003; 11(5):335-342 )。退化性關節炎 (Osteoarthritis)發生的起因可能只是由於輕微損傷，而導致關節軟 骨間質與生長因子分泌兩者之間不分衡，所表現出來的一種現 象。於醫學上，對於退化性關節炎的處理方式多是給予所謂的緩 和劑，其目的在於減輕疼痛，這些藥劑包括對乙醯基胺基酚 (acetaminoptoi)、非類固醇消炎藥(nonster〇idal antiinflammatory =mgs)、f 質類固醇(corticoster〇ids)及透明質酸(hyalur〇nic 北叫 等。目刖於醫療上，葡萄醣胺(glue〇samine)及硫酸軟骨素 (chondroitm sulfate’簡稱CS)被發現可以作為所謂的軟骨保護劑， 用於治療退化性關節炎。這-類化合物係被當作食品添加劑行銷 於美國，但是在歐洲則將其歸類為藥物。 硫酸，骨素是醣胺素glycosamin〇glycans (GAGs)的主要成分 之一 ’以葡萄醣胺(glUC0samine)雙糖為單體聚合而成。目前認為， 於曰常食物中添加此類化合物，可以刺激軟骨再生（c〇nteA等人 如财滅哮缝1995; 45⑻:918_925)。例如，McAlind〇n 人，臏關節與膝關節的退化性關節炎病人身上，使用葡萄膽胺 (glucosamme )及硫酸軟骨素進行治療退化性關節炎，並進行了系 統性的分析，其結絲明，使㈣萄_(§1職—e)及硫酸軟 201113024 月素，於0A治療有一定的益處（c〇r(J〇ba F，跑以施·，湖：及 〇 L” 2003，如前述）。部分證據顯示，葡萄糖胺 和硫酸軟骨素複方製劑的活性與其結構式上的硫 有°基於此硫酸軟骨素在退化性關節炎上對於治療效果有 ϋϊίϋί獻，硫酸軟骨素可幫助維持關節的柔軟與_性， ，進關即軟月的修復。因為在退化性關節炎發生時，軟骨細胞會 3分解酵素代謝軟骨’造雜織的傷害，以及關節滑囊液的不 品質；此外，硫酸軟骨素可抑制彈性蛋白酶(dastase)及透明質酸 S^(hyaluronidase)的作用’同時抑制肥大細胞(mast ceu)造成的發炎 反應，且隨著硫酸軟骨素劑量增加，其抑制效果更顯著，因此推 測硫酸軟骨$是肥大細胞分泌發炎有朗子的有效抑制劑。說明 在退化性關節炎的應用上，硫酸軟骨素除促進軟骨的自我修復 外，也可幫助減緩發炎反應。Bassleer等學者亦在體外實驗結果 發現，硫酸軟骨素可以刺激體外培養的軟骨細胞中pr〇te〇glycans 的合成，顯示硫酸軟骨素對於GAGs的代謝具有正面調控的能力 (BassleerC.等人 咖心似 1992; 14(5):231-241;及Karzel K.與 Lee KJ. 则加/. 1982; 41(5):212-218 )。 軟骨組織由於缺少神經及血管的分佈，因而導致在疾病發生 上（例如’退化性關節炎）的自我修復不易，以細胞為主之治療 法，是目前用於治療關節疾病的一個趨勢。但是，倘若將軟骨細 胞於體外進行增生，將會發生去分化現象，也就是，軟骨細胞於 體外會喪失其表現形態（phenotype)’因而在臨床應用上會有細胞 數量不足的問題（參見，例如’Brittberg M.等人iV五叹/«/Mei/1994; 331:889—95，Temenoff JS 與 Mikos AG.所omaien’a/s 2000; 21:431-40 ; SolchagaLA. C/h Ο—p 及e/加及烈2001: S161-70 ;及201113024 VI. OBJECTS OF THE INVENTION: TECHNICAL FIELD The present invention relates to the use of chondroitin sulfate to promote bone marrow cell differentiation, and more particularly to the use of radiation-treated chondroitin sulfate for promoting bone marrow cell differentiation. [Prior Art] In recent years, 'the literature has suggested that chondroitin sulfate in glycosaminoglycans (GAGs) may have the potential to treat degenerative arthritis (see, for example, CordobaF, NimniME. 0 you (10) 呦rto 2003; 11 (3): 228-230; and Lippiello L. (3⁄4 plus 2003; 11(5): 335-342). The cause of degenerative arthritis (Osteoarthritis) may be due to minor damage, resulting in articular cartilage interstitial and growth factors. A phenomenon that is manifested by the imbalance between the two. In medicine, the treatment of degenerative arthritis is mostly given by a so-called palliative, which aims to alleviate pain. These agents include acetaminophen. Acetaminoptoi, nonster 〇idal antiinflammatory (mgs), f steroids (corticoster〇ids), and hyaluronic acid (hyalur〇nic north called etc.. See medical treatment, glucosamine (glue〇 Samine) and chondroitm sulfate (CS) have been found to be used as so-called chondroprotective agents for the treatment of degenerative arthritis. These compounds are marketed as food additives. Country, but in Europe it is classified as a drug. Sulfuric acid, osteogen is one of the main components of glycosamine glycosamin〇glycans (GAGs) 'polymerized with glucosamine (glUC0samine) disaccharide as monomer. Adding such compounds to Yu's food can stimulate cartilage regeneration (c〇nteA et al., et al. 1995; 45(8): 918_925). For example, McAlind〇n, ankle joint and knee joint degenerative arthritis On the patient, glucosamme and chondroitin sulfate were used to treat degenerative arthritis, and a systematic analysis was carried out. The knot was clear, so that (4) _ (§1 job-e) and sulphuric acid soft 201113024 month There is a certain benefit in the treatment of 0A (c〇r (J〇ba F, running to Shi, Lake: and 〇L) 2003, as mentioned above). Part of the evidence shows that the activity of glucosamine and chondroitin sulfate combination preparation Compared with the sulfur in its structural formula, chondroitin sulfate can help maintain the softness and _ sex of the joint, based on the fact that this chondroitin sulfate is effective in the treatment of degenerative arthritis. Sexual joint When it occurs, the chondrocytes will decompose the enzyme to metabolize the cartilage's damage, and the quality of the joint synovial fluid; in addition, chondroitin sulfate can inhibit the action of elastase and hyaluronidase. 'At the same time inhibiting the inflammatory response caused by mast ceu (mast ceu), and with the increase in the dose of chondroitin sulfate, the inhibitory effect is more significant, so it is speculated that the sulfated cartilage $ is an effective inhibitor of mast cells secreting inflammation and Langzi. Description In the application of degenerative arthritis, chondroitin sulfate can also help to slow down the inflammatory response in addition to promoting the self-repair of cartilage. Bassleer et al. also found in vitro that chondroitin sulfate can stimulate the synthesis of pr〇te〇glycans in chondrocytes cultured in vitro, indicating that chondroitin sulfate has a positive regulatory ability for the metabolism of GAGs (Bassleer C. et al. 1992; 14(5): 231-241; and Karzel K. and Lee KJ. ADD/. 1982; 41(5): 212-218). Due to the lack of distribution of nerves and blood vessels, cartilage tissue is difficult to self-repair in the occurrence of diseases (such as 'degenerative arthritis), and cell-based treatment is a trend currently used to treat joint diseases. However, if the chondrocytes are proliferated in vitro, dedifferentiation will occur, that is, the chondrocytes will lose their phenotype in vitro' and thus there will be a problem of insufficient cell number in clinical applications (see, for example, 'Brittberg M. et al. iV sigh/«/Mei/1994; 331:889-95, Temenoff JS and Mikos AG. omaien'a/s 2000; 21:431-40; SolchagaLA. C/h Ο-p And e/plus and Lie 2001: S161-70; and
Homicz MR 等人 Otolaryngol Head Neck Surg 2002; 127:398-408)。近年來，由於幹細胞具有兩種特性：多功能性 (multi-potency)與自我更新能力(seif-renewal)，故被認為可以分化 成多種類的細胞。因此若將其應用於軟骨組織重建工程上，除了 能克服細胞數不足的問題外，更可以被誘導分化成軟骨細胞 201113024 (Fehrer C·與 Lepperdinger G. 2005; 40:926-30.Homicz MR et al. Otolaryngol Head Neck Surg 2002; 127:398-408). In recent years, stem cells are thought to be able to differentiate into a wide variety of cells because of their two characteristics: multi-potency and seif-renewal. Therefore, if it is applied to cartilage tissue reconstruction engineering, in addition to overcoming the problem of insufficient cell number, it can be induced to differentiate into chondrocytes 201113024 (Fehrer C· and Lepperdinger G. 2005; 40:926-30.
Thomson JA.等人 5W⑼ce 1998; 282:1145-7 )〇 【發明内容】 因此，本發明之一方面係關於，一種於活體外促進骨髓細胞 分化為軟骨細胞之方法’其特徵在於將骨髓細胞培養於添加有硫 酸軟骨素之培養基t。於一項具體態樣，所添加之硫酸軟骨素為 經輻射線處理之硫酸軟骨素。於另於一項具體態樣，所添加之硫 響 酸軟骨素為食品級硫酸軟骨素。 本發明之另一方面係關於，一種用於活體外誘導骨髓細胞分 化為軟骨細胞之培養基’其特徵在於包含硫酸軟骨素及供骨髓細 ，生長之培養基。於一項具體態樣，該培養基中所包含之硫酸軟 骨素為經輻射線處理之硫酸軟骨素。於另於一項具體態樣，該培 養基中所包含之硫酸軟骨素為食品級硫酸軟骨素。於又另於一項 具體態樣，該培養基為無血清培養基。 【實施方式】 鲁 硫I軟骨素為主要以雙糖單體[glucuronic acid (glcUA) β ^3Y_aeetylgalaetQSamine(galNAc)]所組合而成的聚合物，其依照 /、反醋上不同的硫化型態分為：硫酸軟骨素A (type a硫酸軟骨 素’ chondroit；in-4-sulfate)及硫酸軟骨素c (type C硫酸軟骨素， ehondiOitin-6-sulfate)(參見，RajQan r 等人 c/zem 5ζ·ο/· 2005; 12(3):267-277) ’彼等之結構式分別如圖1(A)及1(B)所示。於人類 =長中^關節軟骨組織中發現，硫酸軟骨素c及硫酸軟骨素A兩 ^以等量鍵結於聚集蛋白聚糖(Aggrecan)上’共有約30〜40雙糖個 =體’而於成熟關節軟骨中，則由约20雙糖個單體組成聚集蛋白 &糖i其中大部份為硫酸軟骨素c。 軟骨細胞有兩種主要的細胞外間質--膠原蛋白二型(Collagen 201113024 type II)以及大量的醣胺素，其中膠原蛋白二型係提供機械力上的 穩定性’而各種類醣胺素（包含硫酸軟骨素'透明質酸等）則鍵 結在稱為聚集蛋白聚糖(Aggrecan)的核心蛋白上，以便形成大分子 的肽聚醣。 於本發明之一項具體態樣，係藉由在膠原蛋白二型(c〇L2)的 微環境之下’探討食品級硫酸軟骨素對於兔子骨髓細胞分化為軟 骨細胞的影響。於該實驗令，係使用Real_Time PCR分析二種細 胞外間質基因：膠原蛋白二型基因與聚集蛋白聚糖基因，及一種 管豕基因（housekeeping gene)甘油駿-3-雄酸脫氫酶 (glycemldehyde-3-phosphate dehydrogenase ’ GAPDH)基因之表 現，來評估軟骨細胞於C0L 2支架中的不同分化狀態。 實施例 以下，本發明將詳細描述特殊的具體實施例。這些具體實施 例經由發明解釋提供，並非意欲用以限制本發明。在發明的範園 及精神内，本發明存在傾向於包括這些及其他變更及變動。 實施例上硫酸軟骨素促進料賴細胞分化絲骨細胞 本實驗係將兔子骨髓細胞培養在膠原蛋白二型的微環境之 下二夕卜加以未經或經不同輕射強度處理之硫酸軟骨素之無血清培 12 fGI!_b3生長因子）的培養條件下，觀察對於骨髓細胞在 ‘二μj的微％境下培養誘導分化之影響，並以所選擇標的 基因（膠原蛋白type Π基因與聚集蛋白聚糖基因）之表現，作為 評估兔子倾細胞能否分化為軟骨細胞的指標。 a. _膠原蛋白二型支架之绪☆ 蓉人^蛋白ί敎架之製備，主要是參考之前實驗㈣法（K〇 白^的支_—_萃取，較分離 乾備用。取勝原蛋白刚mg溶於10 ml 0 5Μ醋酸中，擾摔均句， 201113024 將溶液置於塑膠模具（直徑8-mm，深度5-mm)中，於-2(TC冰;東 成型後，凍乾，將支架浸泡於70%乙醇/PBS溶液中，直至pH值 達7.0，隨後以4.4 mM的梔子平(genipin)進行交聯48小時，同樣 以70%乙醇/PBS溶液將交聯劑清洗乾淨’並置於70%乙醇中^ 菌保存。 < b.兔子骨髓細胞的培卷 六個月大2.5公斤紐西蘭白兔，以Zoletil5〇 (l〇_i5mg/kg)及 Xylazine (5mg/kg)進行肌肉内（IM)注射達到麻醉目的。於兔 子後肢膝蓋部位進行剃毛及消毒，以18號針頭抽取約2 ml骨趙^ 隨後將抗凝血劑肝素(heparin) (3000單位/ml)打入，並混合均句 防止血液凝固。之後以PBS沖洗兩次，將脂肪部分去除後離心， 以MSCs增生培養液（含有α_ΜΕΜ (最低極限必須培養基 (Minimum Essential Medium alpha-modified)，Hyclone)為基礎， 額外添加 20% (v/v) FBS，Gibco、4 ng/ml b-FGF (basic fibroblast growth factor-CytoLab/PeproTech Asia ’ Israel)、100 u/ml 青霉素、 100 ug/ml鏈霉素、0.025 ug/ml兩性霉素B、與1.5 mg/ini碳酸氫 鈉，Gibco)培養，並將其放置於3rC含有5% c〇2之培養箱中培 養，五天後更換培養基’待當其生長至滿盤（c〇n£|uence)時，須 進行繼代、保存或進行實驗。 、 為降低血清對骨髓細胞分化的影響，遂於進行誘導時改以無 血清培養基（DMEM (hyclone)、50 ug/ml Ascorbic acid (Sigma)、 0.4 mM L-脯胺酸（Sigma)、Η)·7 M Dexamethasone (Sigma)、l〇 ng/ml TGF-b3 (CytoLab/PeproTech Asia ’ Israel)、100 Ug/mi 丙酮酸 鈉(Sigma)、1%ITS+1 (sigma)、lOOU/ml 青霉素、i〇〇ug/ml鏈霉 素。、0.〇25 ug/ml兩性霉素b、與i.5 mg/ml碳酸氫鈉〕，培養在 37 C及5% C〇2培養箱中培養。本實驗係將未經輻射處理，及經輻 射線處理過後之食用級硫酸軟骨素以1〇 ug/ml,加至培養基申， 分別觀f其對於骨髓細胞分化表現的影響。將細胞以2χ1〇6個細 胞/ml，藉由針筒注射的方式注射至如前述製備之膠原蛋白二型支 201113024 c.龜酸軟骨素之輻射绫虛揮 將食品級硫酸軟骨素分別以5 kgy、1G kgy、15 kgy及2〇 kgy 之輻射線照射’而得食1、食2、食3與食4等樣本。其中，所得 ^本含有分子量為4,519與2,424道耳頓之硫酸軟骨素，食 樣本s有分子量為5,389與2,886道耳頓之硫酸軟骨素，食3樣 $要含有分子量為5,764與3，G92道耳頓之硫酸軟骨素，而食4 樣本主要含有分子量為5,957與3,23〇道耳頓之硫酸軟骨素。 d.摇的基因表現分析(Real-Time ΡΓ.Ρ丨 為了獲得軟骨細胞於COL 2支架中不同分化狀態，因此使用 =錄酶(、ReV跡Transeriptase) PCR _行町三^細胞外間 質土 的分析：collagen type Η、aggrecan 及管家基因 GApDH 基 ，。首，，將欲進行實驗之細胞加入i mlTri_Reagent⑧（Gib⑺）， f置於室溫下侧15min。將溶液吸出轉移至無菌之微量離心管 中，並加入0.2 ml氯仿（Sigma)，經震盪混合並靜置於室溫下2 〜15 mm後，以12000印爪於4。〇下離心15 min。離心後，取出 水相層加入0.5 mi異丙醇（sigma)，均勻混合後靜置室溫5〜 10 min。以-於代下離心15 min後曼再 。以1 ml75%乙醇（Sigma)清洗RNA沈澱。利用7500rpm於4 ^離、5min，移除上清液，並將所得之沈澱加以風乾，以 EPC/H20將其復溶後，存放於待用。 取出 1 Mg總體RNA 以 MMLV反轉錄酶（revefset]ransei>iptase) 輔以ohgo-dT引子（Fermantas)，將其反轉錄為cDNA。本實驗 係採用相對定量法，以細胞内GAPDH基因表現做為參照，將每 個樣本中之待測基因的含量標準化（相對定量以Ct差異代表基因 表現量相差之倍數，其詳細計算方式請參考美/國Applied 201113024Thomson JA. et al. 5W(9)ce 1998; 282:1145-7) [Invention] Accordingly, one aspect of the present invention relates to a method for promoting differentiation of bone marrow cells into chondrocytes in vitro, which is characterized by culturing bone marrow cells The medium t to which chondroitin sulfate is added. In one embodiment, the added chondroitin sulfate is a radiation treated chondroitin sulfate. In another embodiment, the added chondroitin chondroitin is a food grade chondroitin sulfate. Another aspect of the present invention relates to a medium for in vitro induction of differentiation of bone marrow cells into chondrocytes, which is characterized by comprising chondroitin sulfate and a medium for finening and growing bone marrow. In one embodiment, the sulphuric acid sulphate contained in the medium is a radiation treated chondroitin sulfate. In another embodiment, the chondroitin sulfate contained in the medium is a food grade chondroitin sulfate. In yet another embodiment, the medium is a serum-free medium. [Examples] Lusulfur I chondroitin is a polymer mainly composed of glucuronic acid (glcUA) β ^3Y_aeetylgalaet QSamine (galNAc), which is different in vulcanization type according to /, vinegar. For: chondroitin sulfate A (type a chondroit; chondroit; in-4-sulfate) and chondroitin sulfate c (type C chondroitin sulfate, ehondiOitin-6-sulfate) (see, RajQan r et al. c/zem 5ζ) ·ο/· 2005; 12(3):267-277) 'The structural formulas of these are shown in Figures 1(A) and 1(B) respectively. In human = long middle ^ articular cartilage tissue, it was found that chondroitin sulfate c and chondroitin sulfate A were equally bonded to aggrecan (Aggrecan), which had a total of about 30 to 40 disaccharide = body. In mature articular cartilage, aggregate protein & sugar i is composed of about 20 disaccharide monomers, most of which is chondroitin sulfate c. Chondrocytes have two major extracellular mesenchymes - Collagen type 2 (Collagen 201113024 type II) and a large number of glycosaminoglycans, of which collagen type II provides mechanical stability' and various classes of glycosides (Containing chondroitin sulfate 'hyaluronic acid, etc.) is bonded to a core protein called aggrecan to form a macromolecular peptidoglycan. In one embodiment of the invention, the effect of food grade chondroitin sulfate on the differentiation of rabbit bone marrow cells into soft bone cells is explored by the microenvironment of collagen type 2 (c〇L2). In this experimental procedure, Real_Time PCR was used to analyze two extracellular mesenchymal genes: a collagen type 2 gene and an aggrecan gene, and a housekeeping gene glycerol-androgen-3-dehydrogenase ( The expression of the glycemldehyde-3-phosphate dehydrogenase 'GAPDH' gene was used to assess the different differentiation status of chondrocytes in the C0L 2 scaffold. EXAMPLES Hereinafter, the present invention will be described in detail with reference to specific embodiments. These specific examples are provided by way of illustration of the invention and are not intended to limit the invention. The present invention is intended to embrace these and other changes and modifications. In the embodiment, chondroitin sulfate promotes cell differentiation of silk fibroblasts. In this experiment, rabbit bone marrow cells are cultured under the microenvironment of collagen type II, and chondroitin sulfate is treated without or with different light intensity. Under the culture conditions of serum-free culture 12 fGI!_b3 growth factor, the effect of bone marrow cells on the differentiation induced by culture in the micro-% of 'μμj was observed, and the selected target gene (collagen type Π gene and aggregate protein were aggregated). The expression of the glycogene is used as an indicator for assessing whether rabbit pour cells can differentiate into chondrocytes. a. _ Collagen type 2 stent ☆ 蓉人^ protein 敎 之 frame preparation, mainly refer to the previous experiment (four) method (K 〇 white ^ branch ___ extraction, more separation dry spare. Winning protein just mg Dissolved in 10 ml 0 5 Μ acetic acid, disturbed, sentence, 201113024 Place the solution in a plastic mold (diameter 8-mm, depth 5-mm), at -2 (TC ice; after forming, freeze-dry, bracket Soaked in 70% ethanol / PBS solution until the pH reached 7.0, then cross-linked with 4.4 mM genipin for 48 hours, also washed the cross-linking agent in 70% ethanol / PBS solution 'and placed in 70 % ethanol in the preservation of bacteria. < b. rabbit bone marrow cells cultured six months old 2.5 kg New Zealand white rabbit, Zoletil5〇 (l〇_i5mg/kg) and Xylazine (5mg/kg) for intramuscular (IM) injection for anesthesia purposes. Shaving and disinfecting the knees of the hind limbs of the rabbits, taking about 2 ml of bone with an 18-gauge needle and then injecting the anticoagulant heparin (3000 units/ml). The mixed average sentence prevents blood coagulation. After that, it is washed twice with PBS, and the fat is partially removed and then centrifuged to grow MSCs (containing α_Μ). Based on M (Minimum Essential Medium alpha-modified, Hyclone), add 20% (v/v) FBS, Gibco, 4 ng/ml b-FGF (basic fibroblast growth factor-CytoLab/PeproTech Asia) 'Israel', 100 u/ml penicillin, 100 ug/ml streptomycin, 0.025 ug/ml amphotericin B, and 1.5 mg/ini sodium bicarbonate, Gibco), and placed in 3rC containing 5% c Cultivate in the incubator of 〇2, and change the medium after five days. When it is grown to full plate (c〇n£|uence), it must be subcultured, preserved or tested. To reduce the differentiation of serum into bone marrow cells. The effect is to change to serum-free medium (DMEM (hyclone), 50 ug/ml Ascorbic acid (Sigma), 0.4 mM L-proline (Sigma), Η) · 7 M Dexamethasone (Sigma), l 〇ng/ml TGF-b3 (CytoLab/PeproTech Asia ' Israel), 100 Ug/mi sodium pyruvate (Sigma), 1% ITS+1 (sigma), lOOU/ml penicillin, i〇〇ug/ml streptomycin . 0. 〇25 ug/ml amphotericin b, and i.5 mg/ml sodium bicarbonate], cultured in 37 C and 5% C〇2 incubator. In this experiment, the edible grade chondroitin sulfate after radiation treatment and radiation treatment was added to the medium at a dose of 1 〇 ug/ml, respectively, and its effect on the differentiation performance of bone marrow cells was observed. The cells were injected at a dose of 2χ1〇6 cells/ml by syringe injection to the collagen type II branch prepared as described above. 201113024 c. The radiation of the chondroitin chondroitin was used to treat the food grade chondroitin sulfate to 5 Gag, 1G kgy, 15 kgy, and 2 〇kgy radiation exposure to get food 1, food 2, food 3 and food 4 samples. Among them, the obtained product contains chondroitin sulfate having a molecular weight of 4,519 and 2,424 Daltons, and the food sample has a molecular weight of 5,389 and 2,886 Daltons of chondroitin sulfate, and the food sample has a molecular weight of 5,764 and 3, G92. Chondroitin sulfate, while the food sample 4 mainly contains chondroitin sulfate with a molecular weight of 5,957 and 3,23 Daltons. d. Shaking gene expression analysis (Real-Time ΡΓ.Ρ丨 In order to obtain different differentiation status of chondrocytes in COL 2 scaffold, use = recording enzyme (, ReV trace Transeriptase) PCR _ Xingmachi three ^ extracellular soil Analysis: collagen type Η, aggrecan and housekeeping gene GApDH base, first, add the cells to be tested to i mlTri_Reagent8 (Gib (7)), f placed at room temperature for 15 min. Transfer the solution to a sterile microcentrifuge tube Add 0.2 ml of chloroform (Sigma), mix with shaking and let stand at room temperature for 2~15 mm, then centrifuge with 12,000 paws for 4 min. Centrifuge for 15 min. After centrifugation, remove the aqueous layer and add 0.5 mi. Isopropanol (sigma), evenly mix and let stand at room temperature for 5~10 min. After centrifugation for 15 min, transfer to RNA. Wash the RNA pellet with 1 ml of 75% ethanol (Sigma) at 4500 rpm. After 5 min, the supernatant was removed, and the resulting pellet was air-dried, reconstituted with EPC/H20, and stored for use. Remove 1 Mg total RNA with MMLV reverse transcriptase (revefset) ransei>iptase) ohgo-dT primer (Fermantas), which is reverse transcribed into cDNA. The system uses the relative quantification method, and uses the intracellular GAPDH gene expression as a reference to standardize the content of the test gene in each sample (relative quantification is a multiple of the Ct difference representing the difference in gene expression, please refer to the US for the detailed calculation method. /国Applied 201113024
Biosystems SDS 1.3.1 軟體）。real-time PCR 則使用 ABI 引子 7300 序列偵測系統（Applied Biosystems)進行反應’並以SYBR Green PCR master mix (Applied Biosystems)作為定量試劑。下表一列示， 於Real-Time PCR中所使用之引子序列。 表一、用於Real-Time PCR之核苦酸引子 基因 引子序列 擴增產物 大小(bp) Aggrecan Fw: TGAGGAGGGCTGGAACAAGTACC Rv: CCACTGGTAGTCTTGGGCATTGT 169 Collagen II Fw: ACTTGCGTCTACCCCAATCC Rv: ACGTTGGCAGTGTTGGGAG 155 GAPDH Fw: AGCCTCAAGATCATCAGCAATG Rv: GGCCATCACGCCACAGTTT 172Biosystems SDS 1.3.1 software). The real-time PCR was performed using the ABI primer 7300 Sequence Detection System (Applied Biosystems) and used as a quantitative reagent by SYBR Green PCR master mix (Applied Biosystems). The primer sequences used in Real-Time PCR are listed in Table 1 below. Table 1. Nuclear acid primer for Real-Time PCR Gene primer sequence Amplification product Size (bp) Aggrecan Fw: TGAGGAGGGCTGGAACAAGTACC Rv: CCACTGGTAGTCTTGGGCATTGT 169 Collagen II Fw: ACTTGCGTCTACCCCAATCC Rv: ACGTTGGCAGTGTTGGGAG 155 GAPDH Fw: AGCCTCAAGATCATCAGCAATG Rv: GGCCATCACGCCACAGTTT 172
G.結果 結果參見圖2及圖3所示，相較於未經處理之硫酸軟骨素而 言，經不同強度輻射線處理之硫酸軟骨素，都加強了 aggrecan'及 CollagenII的基因表現。此外，研究結果發現，隨著不同輻射強度 處理的程度增加，其基因表現亦隨之往上增加，而於第14天(W2) k，研九結果更發現’於處理強度為照射6〇分鐘（此處理條件是 否應改為20 kgy?)之硫酸軟骨素其基因之表現最好哪_及 的基因表現’相較於未經輕射線處理之硫酸軟骨素，在 誘導遺細胞分化為軟骨細胞有更好的表現。此暗示，經處理之 201113024 是，特徵，任何組合方式組合。於 2代特徵，。因此，除非另行清楚 僅為-系列同等物或類似特徵之實例。 不之各特徵 本特易地確定本發明之基 以使其適於各種不咖途與狀泥。‘發與修飾， 含其他具體態樣。 ；申印專利範圍内亦包 201113024 【圖式簡單說明】 圖1(A)與(B)分別列示硫酸軟骨素a (type A碳酸 學名為chondroitin-4-sulfate)及硫酸軟骨素c ( 缺、 素，化學名為chondroitin-6-sulfate)之結構。 瓜-夂人月 圖2列示骨髓細胞培養在膠原蛋白二型的微淨产 以未經處理及經不同轄射強度處理之硫酸軟骨素^，卜，夕卜力口 基因表現的影響，該基因之表現係作為評估骨翁細胞^^= 軟骨細胞的指標。控制組:未添加任何硫酸軟骨匕為 理:未經輻射線處理之硫酸軟骨素，食*1:指經輪射 *2:指經輻射線照射l〇kgy，食*3:指經輻射線昭gy，艮 指經輻射線照射20kgy,縱軸：Fold Difference f、的θ gy，食*4: 組第〇天之細齡現 efenee.心岐她於控制 圖3列示骨髓細胞培養在膠原蛋白 以未經處理及經不_職度處歡贿，外加 基因表現的影響，該基因之表現係 H對於Aggrecan 軟骨細胞触1。食*1:指經輻射線照射° 為 20kgy分鐘，縱軸..F〇ldDilfere / 4.扎經輻射線照射 細胞表現 ence.扣的疋相較於控制組第〇天之 【主要元件符號說明】G. Results The results are shown in Figures 2 and 3. Compared to untreated chondroitin sulfate, chondroitin sulfate treated with different intensity radiation enhanced the gene expression of aggrecan' and CollagenII. In addition, the results of the study showed that as the degree of treatment with different radiation intensity increased, the gene performance increased accordingly. On the 14th day (W2) k, the results of the study were found to be 'in the treatment intensity for 6 minutes. (Whether this treatment condition should be changed to 20 kgy?) The chondroitin sulfate has the best performance of the gene. The gene expression is compared to the untreated chondroitin sulfate, which induces differentiation of the cells into chondrocytes. Have a better performance. This implies that the processed 201113024 is a combination of features and any combination. In the 2nd generation features. Therefore, unless otherwise clear, only examples of -series equivalents or similar features are shown. Various features The base of the present invention is specifically determined to be suitable for a variety of non-sticky and muddy forms. ‘fat and modification, including other specific aspects. The scope of the patent application also covers 201113024 [Simplified illustration] Figure 1 (A) and (B) respectively list chondroitin sulfate a (type A carbonate name chondroitin-4-sulfate) and chondroitin sulfate c ( The structure of the deficiency, prime, chemical name chondroitin-6-sulfate). Figure 2 shows the effect of gene expression of chondroitin sulfate in the bone marrow cell culture on the micro-production of collagen type II, which has been treated untreated and treated with different radiant strengths. The gene expression was used as an indicator for evaluating the bone cells of the bones ^^= chondrocytes. Control group: no added chondroitin sulfate for treatment: chondroitin sulfate without radiation treatment, food *1: refers to the round of injection *2: refers to radiation through the radiation l〇kgy, food *3: refers to the radiation Zhao gy, 艮 refers to radiation radiation 20kgy, vertical axis: Fold Difference f, θ gy, food * 4: group 〇 day of the age of the current efenee. Heart 岐 her control in Figure 3 listed bone marrow cells culture in collagen Proteins are untreated and unpaid, and the expression of this gene is H for Aggrecan chondrocytes. Food *1: refers to radiation radiation ° ° 20kgy minutes, vertical axis..F〇ldDilfere / 4. Ties radiation through the cell performance ence. Deduction of the 疋 phase compared to the control group 〇天之 [main component symbol description 】